Coagulation of Blood Plasma
Blood coagulation is a defensive process in the body, the formation of a fibrin clot being one of its essential components. The fibrin clot is formed as a result of activation of an enzymatic cascade of coagulation factors; many coagulation factors are serine proteases (VIIa, XIIa, XIa, IXa, Xa, thrombin IIa). The enzymatic cascade also involves activated protein C (aPC), a protease activated by thrombin and inactivating coagulation cofactors VIIIa and Va. The cascade is activated by the «extrinsic» pathway for which factor VIIa (fXIIa) and tissue factor (TF, or thromboplastin) are held responsible, or by the «intrinsic» pathway that starts from contact activation of factor XII (fXII, or Hageman factor) (Mann et al. 1990, Blood 76:1-16; Hoffman and Monroe. 2001, Thromb. Haemost. 85:958-965). Contact activation is auto-activation of fXII in case of a blood plasma contact with negatively charged surfaces. fXII is present in blood plasma in its inactive state; when bound with high molecular weight kininogen (HMWK) and plasma prekallikrein, it can be transformed into an active protease, factor XIIa (fXIIa). Any foreign surface, including walls of plastic or glass tubes, stent or catheter walls, etc., can provoke activation of fXII. fXIIa provides proteolytic activation of prekallikrein into its active form, kallikrein, an enzyme responsible, among others, for fibrinolysis system activation, and of the «intrinsic» pathway factor XI into its active form, factor XIa (fXIa) (Ichinose et al. 1986, J. Biol. Chem. 261:3486-3489; Schmaier. 2008, J. Clin. Invest. 118:3006-3009).
Contact Activation Suppression
Suppression of the contact-activated coagulation, which appears in case of contact of a blood or plasma sample (whole blood or blood plasma, including platelet-rich, platelet-poor, or platelet-free plasma) with a foreign surface and prevents the use of the sample for storage, transfusion, or research, is an important issue (Sperling et al. 2009, Biomaterials 30:4447-4456; Streller et al. 2003, J. Biomed. Mater. Res. B Appl. Biomater. 66:379-390). fXIIa is activated in case of blood collection from the circulation, for example: 1) during blood collection into a tube, from the surfaces of needles, ducts, or tube walls, 2) during storage of frozen blood plasma and subsequent plasma thawing, or 3) when using artificial (extracorporeal) blood circulation apparatus, in particular during blood or plasma transfusion. Contact activation during blood collection or thawing of frozen plasma can initiate clotting in the tube, thus preventing the sample processing and coagulation research (Smith et al. 2010, Blood Coagul. Fibrinolysis. 21:692-702; Suontaka et al. 2005, Vox. Sang. 88:172-180). During autotransfusion or repeated blood infusion, the contact pathway activation leads to increased risk of thrombosis. To block blood clotting when using the artificial blood circulation apparatus, the ducts and other surfaces of the apparatus are covered with fXa or thrombin inhibitors, such as heparin, benzamidine, etc. (Hsu. 2001, Perfusion 16:417-428; Gouzy et al. 2006, Biointerphases 1:146-155). The disadvantage of such method of coagulation blocking is that the addition of fXa and thrombin inhibitors does not prevent generation of fXIIa which can initiate clotting after infusion of blood or plasma into the circulation.
The most common way of suppressing coagulation during blood collection is provided by preliminary addition of calcium ions chelators (for example, of sodium citrate, EDTA, etc.) into the collection tube. Chelation of calcium ions blocks membrane-dependent reactions of fX and thrombin activation (Fischer. 2007, Hemodial. Int. 11:178-189). During the coagulation testing, a coagulation activator (in particular, TF or the contact pathway activator) is added into the test sample containing a chelator, and the plasma is recalcified (by adding an excess amount of calcium ions), after which the fibrin clot formation parameters are studied. The method of coagulation suppression by chelator addition shows a number of disadvantages. The article Mann et al. 2007, J. Thromb. Haemost. 5:2055-2061 demonstrated that chelation of calcium ions during blood collection and subsequent sample recalcification modify the dynamics of fibrin clot formation, formation of thrombin, and aggregation of platelets, in comparison with the samples without addition of the chelator. Chelator addition does not suppress activation of fXI and fIX initiated by fXIIa which is formed during blood collection and sample storage (Nossel et al. 1968, J. Clin. Invest. 47:1172-1180; Oller et at 1976, J. Surg. Res. 20:333-340). Thus, when testing the coagulation, in particular the TF-initiated coagulation through the «extrinsic» pathway (considered to be physiological and responsible for the coagulation in the body in normal conditions), the generated fXIIa also provokes coagulation by the contact pathway, introducing unwanted distortions of the research. The contact pathway of activation in such a research is an artifact that can prevent adequate assessment of physiological coagulation effects, lead to increased error and non-conformity of the research results, as well as to the loss of sensitivity to pathological states of coagulation. To suppress the contact activation, avoid appearance of artifacts, and improve the quality of coagulation research, it is necessary to use fXIIa inhibitor during blood collection or preparation of plasma (Luddington and Baglin. 2004, J. Thromb. Haemost. 2:1954-1959). The used contact activation inhibitor must be highly selective: the inhibitor has to lack any impact on the TF-initiated fibrin clot formation and any inhibitory activity against fXIa, fIXa, fXa, and others, at the concentration of the inhibitor in the sample being sufficient for efficient suppression of the contact pathway of coagulation.
Known Contact Activation Inhibitors
A number of fXIIa inhibitors is known in the field, in particular plasma coagulation inhibitors, such as C1-inhibitor or alpha2-macroglobulin (Davis et al. 2008, Mol. Immunol. 45:4057-4063). Protein inhibitors of trypsin which inhibit fXIIa are also well known in the field; these inhibitors were isolated from different organisms, such as bacterium E. coli (Ulmer et al. 1995, FEBS Lett. 365:159-163), or plant seeds (Wynn and Laskowski. 1990, Biochem. Biophys. Res. Commun. 166:1406-1410). The inhibitors isolated from plant seeds include corn trypsin inhibitor, or CTI (Hojima et al. 1980, Thromb. Res. 20:149-162), as well as inhibitors from the squash family: Cucurbita maxima trypsin inhibitor, CMTI-III (Krishnamoorthi et al. 1990, FEBS Lett. 273:163-167), Luffa cylindrica trypsin inhibitor, LCTI-III (Ling et al. 1993, J. Biol. Chem. 268:810-814), and others. Some other non-protein fXIIa inhibitors are known (Woodruff et at 2013, J. Thromb. Haemost. 11:1364-1373). However, most of the said inhibitors are non-selective: besides fXIIa, they also inhibit other coagulation factors, making it impossible to use them in the research of fibrin clot formation.
CTI is believed to be the most selective of all existing inhibitors. CTI has been used for inhibiting contact activation in the assays of TF-initiated coagulation in a sample of blood or its component (U.S. Pat. No. 6,403,381, cl. G01N33/86, publ. Jun. 11, 2002), particularly in the global assays of hemostasis, such as thrombin generation test. However, the data regarding insufficient selectivity of CTI, its influence on the blood coagulation system and other systems interacting with coagulation, are known. For example, CTI can delay fibrinolysis, supposedly by inhibiting the tissue activator of plasminogen (Nielsen. 2009, Blood Coagul. Fibrinolysis 20:191-196).
A fXIIa inhibitor infestin-4 (domain 4 of the protein infestin from the midgut of a blood-sucking insect Triatoma infestans) that belongs to the family of Kazal-type inhibitors is also known in the field (Campos et al. 2004, FEBS Lett. 577:512-516). The infestin-4 (Inf4) amino acid sequence and X-ray structure are deposited in the Protein Data Bank, reference number 2ERW.
Known Use of Infestin-4 and Disadvantages of Existing Versions of Infestin-4
The article Hagedorn, Schmidbauer et al. 2010, Circulation 121:1510-1517 demonstrated that infestin-4 is a selective fXIIa inhibitor that blocks fXIIa activity in the thrombosis model in vivo and prevents vascular occlusion without affecting hemostasis parameters. The use of infestin-4 as a fusion protein with albumin in treatment and prevention of thrombosis-related diseases was disclosed in U.S. Pat. No. 8,283,319, cl. A61K 38/16, publ. Oct. 9, 2012. However, the disadvantage of infestin-4 is its inhibitory activity against fXa (inhibition constant Ki 53 nM).
Infestin-4 mutant Inf4Mut15 (Mut15) known in the field has an increased selectivity to fXIIa as it does not inhibit fXa (Campos, Souza et al. 2012, Acta Cryst. D68:695-702). The said mutant was selected as the closest analogue of the present invention. From the prior art, we do not know about inhibition selectivity of fXIIa by the said mutant, that is, we do not know if it possesses any inhibitory activity against fXIa, fIXa, fVIIa, thrombin, or aPC. The use of Mut15, among others in the kit for diagnostics of hemorrhagic and thrombolytic diseases by means of measurement of fXIIa concentration in the patient's blood, is known from patent BRPI 0602496-3, cl. A61 38/36, publ. Feb. 26, 2008, though the said use is not related with diagnostics of diseases by means of measurement of coagulation parameters. Therefore, the high-selectivity fXIIa inhibitor that blocks contact activation during the assay of TF-initiated blood coagulation is not known in the field.
The objective of this invention is to design a high-selectivity fXIIa inhibitor to block contact activation during the assay of blood or plasma coagulation, in particular TF-initiated, as well as during blood or plasma collection and storage.